The overall goal of the proposed research is to determine how the structure of the ubiquitous intracellular calcium receptor, calmodulin, dictates function. The long term objectives are to design inhibitors that will be specific for two calmodulin dependent enzymes, smooth muscle myosin light chain kinase (smMLCK) and the multifunctional calmodulin kinase (CaM kinase). One aim is to utilize our current knowledge of the domain organization and regulation of the CaM kinases to produce chimeric enzymes designed to exhibit altered requirements for calmodulin as a regulatory subunit as well as molecular modes of autoinhibition and activation. The approach is to exchange autophosphorylation/autoregulatory domains, calmodulin binding domains and association domains, express the proteins in the baculovirus system, purify the enzymes and determine their properties. This will proceed in parallel with attempts to determine the 3D structures of purified calmodulin dependent fragments of SmMLCK and CaM kinase. Together the information obtained should aid development of specific enzyme inhibitors as, for example, to serve as smooth muscle specific relaxing agents. The second aim is to map the amino acids on calmodulin that are required to execute an essential role of this regulatory protein in the genetically tractable filamentous fungus, Aspergillus nidulans. In all cells examined to date including from human tissues, calmodulin is required for cell proliferation and is specifically needed for cells to pass from G2 to mitosis. A strain of A. nidulans called AlcCaM has been constructed that is conditional for expression of calmodulin. The strategy is to randomly mutate individual domains of calmodulin and prepare cDNA libraries. The libraries will be placed into the AlcCaM/T23 strain of A. nidulans which is conditional for expression of CaM and harbors a temperature sensitive mutation in the NIMTcdc25 gene such that cells are blocked in G2 at the restrictive temperature. We will evaluate the ability of the mutants to promote the G2/M transition upon release of the t-s block. Rescue will be determined in the absence, at optimal levels and at supraphysiological levels of extracellular Ca2+. The mutant proteins will be produced in bacteria and assayed for Ca2+ binding, enzyme binding using the two hybrid system and enzyme activation properties. Such a screen should identify: a) amino acids required for G2/M; b) proteins that can function in the absence of Ca2+ binding; c) proteins with altered affinity of individual Ca2+ binding loops; and d) mutations that selectively affect sequence specific binding versus activation of a battery of target enzymes.